Method and System for Preventing Migraine Headaches, Cluster Headaches and Dizziness

ABSTRACT

A method and system for addressing the avoidance of migraines, cluster headaches and dizziness by adjusting an individual&#39;s microbiome, and in particular, to the provision of beneficial oral and gut microbes at particular times to enhance a person&#39;s oral health, including through the use of oral strips that adhere to surfaces in the oral cavity.

RELATED APPLICATIONS

This application is a non-provisional of U.S. Provisional PatentApplication Ser. No. 62/387,405, filed on Dec. 24, 2015.

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 14/752,192 filed Jun. 26, 2015, which is acontinuation-in-part application of U.S. patent application Ser. No.14/225,503 filed Mar. 26, 2014, (now issued U.S. Pat. No. 9,445,936,issued Sep. 20, 2016), which is a continuation of U.S. patentapplication Ser. No. 13/367,052, filed Feb. 6, 2012 (now issued U.S.Pat. No. 8,701,671, issuing on Apr. 22, 2014), which claims priority ofU.S. Provisional Patent Application Ser. No. 61/439,652, filed on Feb.4, 2011 and U.S. Provisional Patent Application Ser. No. 61/556,023,filed on Nov. 4, 2011.

This application also is a continuation-in-part application of U.S.patent application Ser. No. 15/270,034, filed Sep. 20, 2016, which is acontinuation-in-part application of U.S. patent application Ser. No.14/954,074, filed on Nov. 30, 2015 (now issued U.S. Pat. No. 9,457,077,issuing on Oct. 4, 2016), which is a continuation-in-part application ofU.S. patent application Ser. No. 14/574,517, filed on Dec. 18, 2014 (nowissued U.S. Pat. No. 9,408,880, issuing on Aug. 9, 2016), which claimspriority of U.S. Provisional Patent Application Ser. Nos. 62/072,476,filed on Oct. 30, 2014; 62/053,926, filed on Sep. 23, 2014; 62/014,855,filed on Jun. 20, 2014; and 61/919,297, filed on Dec. 20, 2013.

This application is also a continuation-in-part application of U.S.patent application Ser. No. 15/228,454, filed Aug. 4, 2016.

This application claims priority of U.S. Provisional Patent ApplicationSer. Nos. 62/387,404, filed Dec. 24, 2015; 62/274,550, filed Jan. 5,2016; and 62/275,341, filed Jan. 6, 2016.

This application is also a continuation-in-part application of U.S.patent application Ser. No. 14/611,458, filed Feb. 2, 2015, which is acontinuation-in-part application of U.S. patent application Ser. No.14/502,097, filed Sep. 30, 2014 (now issued U.S. Pat. No. 9,010,340,issuing on Apr. 21, 2015), which is a continuation of U.S. patentapplication Ser. No. 14,307,651, filed on Jun. 18, 2014 (now issued U.S.Pat. No. 8,936,030, issuing Jan. 20, 2015), which is acontinuation-in-part application of U.S. patent application Ser. No.14/079,054, filed Nov. 13, 2013 (now issued U.S. Pat. No. 8,757,173,issuing on Jun. 24, 2014), which is a continuation of U.S. patentapplication Ser. No. 13/425,913. filed Mar. 21, 2012 (now issued U.S.Pat. No. 8,584,685, issuing on Nov. 19, 2013), and claims priority ofU.S. Provisional Patent Application Ser. No. 61/467,767, filed Mar. 25,2011.

The entire disclosure of the prior applications are considered to bepart of the disclosure of the accompanying application and are herebyincorporated by reference

FIELD OF THE INVENTION

A method and system for addressing the avoidance of migraines, clusterheadaches, and dizziness by adjusting an individual's microbiome, and inparticular to the provision of beneficial oral and gut microbes atparticular times to enhance a person's health.

BACKGROUND OF THE INVENTION

All primary headaches are in need of better treatments. The mostimportant primary headaches (i.e. independent disorders that are notcaused by another disease) are migraine, tension-type headache andcluster headache. Migraine has a prevalence of 10% in the generalpopulation with a lifetime prevalence of 13% in men and 33% in women.Migraine is a highly disabling disease with high personal and socialcosts. To date, the precise mechanisms underlying the pathophysiology ofmigraine have remained elusive. Migraine strikes people during what areexpected to be their most productive years: between ages 20 and 40 formost women, with a slightly higher age range for men. Migraine istypically characterized by unilateral onset of head pain, severeprogressive intensity of pain, throbbing or pounding, and interferencewith the person's routine activities. Accompanying symptoms ofphotophobia (sensitivity to light) or phonosensitivity (intolerance tonoise), as well as nausea and/or vomiting, are common, and often leadsto the inability to perform daily tasks. A large portion of people withmigraine often have no accompanying pain, their predominant symptominstead being vertigo (a spinning sensation) or dizziness/disequilibrium(balance loss), mental confusion, disorientation, dysarthria, visualdistortion or altered visual clarity, or extremity paresis. Patientswith migraine associated vertigo (MAV) are often seen by audiologistsand vestibular rehabilitation therapists for evaluation and treatment.Because the exact mechanisms of migraine are still not completelyunderstood, the management of migraine dizziness presently includes acombination of medications, vestibular rehabilitation, and lifestylemodifications that include limitation of risk factors associated withmigraine (those related to diet, sleep, stress, exercise, andenvironmental factors).

Migraine is a disease associated with increased synthesis and release ofcalcitonin gene related peptide (CGRP) and a migraine attack can beblocked with CGRP antagonists. The actual pain is generated bynociceptors of trigeminal nerve endings in the dura. Low serotoninlevels may sensitize the nociceptors of trigeminal neurons. Triptans andergotamins, which decrease serotonin, are associated with relief ofacute pain. In contrast, tricyclic antidepressants and selectiveserotonin and noradrenaline reuptake inhibitors, which are associatedwith increases in serotonin, are utilized for migraine prevention.

Migraine attacks can be triggered by intrinsic cerebral factors (e.g.calcitonin gene related peptide (CGRP) release), nitric oxide liketri-nitroglycerine, corticotrophin releasing hormone (stress),pro-inflammatory cytokines, and degranulation of mast cells located inthe dura. While migraine has a genetic background, twin studies revealthat the cause of a majority of migraines appears to be due toenvironmental factors. The mechanism of triggering migraine is, however,still not understood. The cause of both migraines and chronic dizzinesshas eluded investigators for centuries and it therefore presents a trulylong felt but unsolved mystery as to its causation and treatment.

SUMMARY OF THE INVENTION

One aspect of the present invention is therefore directed to addressingthe causative agents involved in migraines and dizziness, instead ofmerely addressing the symptoms thereof. As can be seen from the priorart as discussed herein, the diagnosis and treatment of both migrainesand dizziness have continued to be directed to symptoms, rather than toan underlying treatable cause. The present invention changes that focusand thus, provides both an understanding of causative agents and howbest to treat individuals so that their maladies are addressed. Thepresent inventors believe that there is an association between theoccurrence of migraines and gastrointestinal (GI) disorders, includingirritable bowel syndrome (IBS). People who regularly experience GIsymptoms have a higher prevalence of headaches, with a strongerassociation with increasing headache frequency.

While not bound by theory, it is believed that the gut microbiota is anindependent factor that contributes to systemic diseases, ofteninvolving the migration of stimulated immune cells, by systemicdiffusion of microbial products or metabolites, or by bacterialtranslocation as a result of decreased intestinal barrier function. Thebrain and the GI tract are strongly connected via neural, endocrine, andimmune pathways. The gut microbiota, as well as the oral microbiota, isassociated with brain functions and neurological diseases like migraine.

Empirical data exists to reveal the association of migraines withdisruptions of a person's microbiome. For example, children with amother with a history of migraine are more likely to have infantilecolic. Children with migraine are more likely to have experiencedinfantile colic compared to controls. Several studies demonstratedsignificant associations between migraine and celiac disease,inflammatory bowel disease, and IBS.

It is believed that the underlying mechanisms of migraine and GIdiseases are both related to increased gut permeability andinflammation. In addition, it is believed that a person's oralmicrobiome is also responsible for migraines. Thus in severalembodiments of the present invention, modification of the oral as wellas (or solely) the gut microbiome is achieved to combat and addressmigraine occurrences.

The biological nitrogen cycle involves step-wise reduction of nitrogenoxides to ammonium salts and oxidation of ammonia back to nitrites andnitrates by plants and bacteria. The salivary bacterial reduction ofnitrate to nitrite has been recognized as an important metabolicconversion in humans. Several enteric bacteria have also shown theability of catalytic reduction of nitrate to ammonia via nitrite duringdissimilatory respiration. Although to date the importance of thispathway in bacterial species colonizing the human intestine has beenlittle studied, the present inventors submit that it is a major factorinvolved in the occurrence of migraines.

In addition to the connection between the gut microbiome and migraines,there is also believed to be a connection with the oral microbiome of anindividual with various health conditions, including an association withdysbiosis of the oral microbiome and migraines.

Many biochemical, pharmacological, neuropathological, and experimentaldata suggest a role of nitric oxide in the pathogenesis of migraine.Nitric oxide (NO) is a very important molecule in the regulation ofcerebral and extra cerebral cranial blood flow and arterial diameters.It is also involved in nociceptive processing. Glyceryl trinitrate(GTN), a pro-drug for NO, causes headache in normal volunteers and a socalled delayed headache that fulfils criteria for migraine without aurain migraine sufferers. One aspect of the present invention is to preventmigraines via the inhibition of NO production; the blockade of steps inthe NO-cGMP pathway; or the scavenging of NO. The pain signalingmolecules, nitric oxide synthase (NOS) and calcitonin gene-relatedpeptide (CGRP) are implicated in the pathophysiology of migraines.

Non-specific NOS inhibition and a specific neuronal NOS inhibitor havebeen found to attenuate neurogenic dural vasodilation. Interestingly,specific inducible and endothelial NOS inhibitors had no effect.Non-vascular inducible NOS inhibitors have been shown unable to abort orprevent migraine. The present inventors submit that this leaves neuronalNOS inhibition and bacterial NOS inhibition (bNOS) as candidates fortherapy, with the latter being considered the primary causative agentsin preventing migraines. Neurons in the trigeminocervical complex arethe major relay neurons and are the neural substrates of head pain. NOproduction in the TCC and potentially other areas of the brain may beinvolved in triggering migraines, and therefore blocking NO productionis believed to be therapeutic.

The nitric oxide synthase inhibitor NG-monomethyi-L-arginine (L-NMMA)may be employed in several embodiments of the present invention.Blockade of nitric oxide synthases (NOS) by L-NMMA effectively treatsattacks of migraine without aura. Similar results have been obtained forchronic tension-type headache and cluster headache.

Inhibition of the breakdown of cGMP also provokes migraine in sufferers,indicating that cGMP is the effector of NO-induced migraine. Severalrelationships exist between NO, calcitonin gene-related peptide andother molecules important in migraine. Also, ion channels, particularlythe K(ATP) channels, are important for the action of NO.

NO donors and their ability to trigger migraine in patients is wellknown and so nitric oxide synthase (NOS) inhibitors have been exploredfor the treatment of migraine. While NO donors are known to causevasodilation, they also cause activation of neurons in thetrigeminocervical complex of the brain that are not vascular related, aswell as other areas of the brain related to migraine. Thus, the presentinventors submit that non-vascular mechanisms are involved and thatnon-vascular NOS inhibitors are efficacious in the treatment ofmigraines.

Nitric oxide synthases (NOSs) are multidomain metalloproteins firstidentified in mammals as being responsible for the synthesis of thewide-spread signaling and protective agent nitric oxide (NO). Nitricoxide synthases are heme-based monooxygenases that oxidize L-arginine tonitric oxide (NO), a signaling molecule and cytotoxic agent in higherorganisms. NO is one of the main inflammatory mediators involved in bothinflammation and angiogenesis . NO can be synthesized by three differentisoforms of NO synthase: neuronal (nNOS), endothelial (eNOS), andinducible (iNOS) synthases. NO production due to cytokine-inducedexpression of inducible nitric oxide synthase (iNOS) is largely involvedin the pathophysiology of inflammation.

Although NOS-like activity has been reported in many bacteria, only afew bacterial homologs of mammalian NOSs (mNOSs) have been characterizedto date. Nitric oxide synthases (NOSs) play an essential role insynthesizing nitric oxide (NO) by oxidizing l-arginine. NO is asignificant mediator in cellular signaling pathways. It serves as acrucial regulator in insulin secretion, vascular tone, peristalsis,angiogenesis, neural development and inflammation. Due to its importantrole, the inhibition of these vital enzymes provides therapeuticapplications that target NOSs.

Over the past 10 years, prokaryotic proteins that are homologous toanimal NOSs have been identified and characterized, both in terms ofenzymology and biological function. In contrast to mNOSs, which possessboth a catalytic and a reductase domain, the bacterial enzymes lackreductase domains and require the supply of suitable reductants toproduce NO. A notable exception is a NOS from a gram-negative bacteriumthat contains a new type of reductase module.

Bacterial NOSs seem to have functions that differ from those of mNOSs,including nitration of different metabolites and protection againstoxidative stress. Bacterial NOSs provide a better understanding of themechanism of NO synthesis and unveil a variety of new functions for NOin microbes.

In one embodiment, the lactic acid bacterium Lactobacillus reuteri isemployed as it is believed that by doing so, one is able to induceoxytocin, preferably in a manner that offers a sustained induction ofoxytocin, unlike the short effects achieved using intranasal oxytocinsprays, etc. Thus one aspect of the present invention relates to theemployment of probiotics-induced oxytocin to reduce migraine symptoms,especially in the form of an oral adhesive strip as further describedherein.

Other embodiments relate to the employment of calcitonin gene-relatedpeptide (CGRP) as a major player in treating migraines. In variousembodiments, CGRP triggers a chain of events such that increased amountsof CGRP released at the start of a migraine sensitize the trigeminalnerve to what are normally innocuous signals, resulting in inflammationin the nerves that is relayed to the brain as a pain signal and in sucha manner, stop headaches from outside the central nervous system,believed to be active on the trigeminal connections into the brainrather than the brain itself. It is further believed that there is oftena fairly massive change in the permeability of the blood-brain barrierduring a migraine attack and may have an effect on certain sections ofthe brainstem that are not believed to be as well protected by theblood-brain barrier. As is the case with autism, it is believed that agut brain inter-relationship dysbiosis is caused by a loweredLactobacillus spp. and decreased number of Clostridium spp. populationsin an individual's gut microbiome. Thus, restoring the gut microbiomewith appropriate levels of particular bacteria species as set forthherein is one aspect of the present invention. In addition, in variousembodiments, the maintenance of desired populations in the gut isachieved via the employment of fibre that is essential for the fosteringof a healthy gut microbiome once established so that the beneficialbacteria have a food source other than the mucosa proteins that protectthe tissue of the gut. In preferred embodiments approximately 6g/fibre/day is administered to an individual as it is believed that bydoing so one is able to increase the numbers of bifidobacteria in thegut microbiome and thus, increase desired fermentation by increasing thenumber and maintenance of both Bifidobacteriaceae and Lactobacteriaceaefamilies. In preferred embodiments, a diets rich in plantpolysaccharides (i.e., fiber) is employed to confer protection againstvarious ailments associated with the gut microbiome dysbiosis, includingnot only migraines, but also cardiovascular disease (CVD). Such benefitsare believed to be derived from the interactions between carbohydratesthat reach the distal gut and microbes via production of short chainfatty acids (SOFA, e.g., acetate, propionate and butyrate). Thus, invarious embodiments, in addition to the administration of beneficialbacteria as described herein wither to the gut or the oral microbiome ofan individual (or both) the consumption of a diet high in fibre isemployed to increase microbiota populations to generate short chainfatty acids (SCFAs) such as acetate, which has a protective role ininflammatory diseases. Such a diet is further believed to attenuate thedevelopment of cardiac fibrosis due to the belief that inflammation isalso implicated in cardiac remodelling. Thus, a high fibre diet,especially one that includes increased levels of acetate, is believed tonot only reduce the incidences of migraine, but to further result insignificantly less cardiac hypertrophy, perivascular and interstitialcardiac fibrosis, and improved cardiac function. It is further believedthat the protective effects of high fibre and acetate are accompanied bya decrease in the ratio of bacteria from the phylum Firmicutes comparedto Bacteroidetes. In addition to the association of the gut and thebrain as it relates to the incidence of migraines, there is also agut-heart connection and by employing similar efforts to address thedysbiosis of an individuals' gut and oral microbiomes, another aspect ofthe present invention is directed to addressing and significantlypreventing the occurrence of heart disease.

In situations where there is insufficient fiber for the beneficialbacteria to consume, the bacteria end up eroding the mucus of the gutand leads to epithelial access by mucosal pathogens. It is believed thatthere is an increase in the amount of calcitonin gene-related peptidereleased at the start of a migraine that sensitizes the trigeminal nerveto what are normally innocuous signals, resulting in inflammation in thenerves that is relayed to the brain as a pain signal. Thus, one aspectof the present invention relates to the ability to affect the trigeminalconnections into the brain, rather than the brain itself, and to do soby modifying the type, number and maintenance of desired bacteria in anindividual's gut and oral microbiome. In addition to migraine headaches,other aspects of the invention are directed more generally to addressingother pain related conditions, including but not limited tofibromyalgia, cluster headaches, etc.

Therefore, certain embodiments of the present invention are directed toa method of reducing the likelihood of migraine headaches by providingto an individual in need thereof a buccal bioadhesive strip, with suchstrip having a first and second side and having a surface comprising apattern defined by a plurality of spaced apart features each having atleast one microscale dimension and having at least one neighboringfeature having a substantially different geometry. Preferably theaverage spacing between adjacent ones of such features is between 0.5and 5 .mu.m. FIG. 2(a)-(d) illustrate some exemplary surfacearchitectural patterns according to the invention.

A bioadhesive is employed that is adapted to bind to a mucosal membranefor at least 1 hour while inside an individual's mouth. Preferably thestrip includes xylitol. In other embodiments, the strip includes anencapsulated feature containing a desired bacteria, preferably selectedfrom the group consisting of Lachnospira, Veillonella, Faecalibacteriumand Rothia. Still other embodiments include an antibody on the strip toa calcitonin gene-related peptide. The strip(s) may further includeLactobacillus spp. Preferably, methods of the present invention furtherinclude administering a diet of at least 6 g/fibre/day to an individualso as to increase the numbers of bifidobacteria in the gut microbiome ofthe individual. Such a diet should preferably include a high fibre dietthat includes acetate, especially in an amount sufficient to decreasethe ratio of bacteria in the individual's oral cavity from the phylumFirmicutes compared to Bacteroidetes. It is preferred to increase thenumber of both Bifidobacteriaceae and Lactobacteriaceae bacteria. Otherembodiments increase the population of desired bacteria by includingEnterococcus faecium on the strip. It is preferred to remove from theoral cavity of the individual gram negative bacteria associated withperiodontitis, and within 2 hours thereof, to provide the individualwith the strip. Still other embodiments include a strip that includesLactobacillus reuteri bacteria to induce a sustained induction ofoxytocin, and providing the individual with an amount of antibioticsufficient to reduce the number of undesired bacteria in the oralcavity. Prior to the use of the strip, an antibiotic selected from thegroup consisting of tetracycline hydrochloride, doxycycline, andminocycline is used to reduce the number of undesired bacteria in theoral cavity. One objective is to retard the growth conditions forspirochetes and P. gingivalis, believed to be associated with migraineheadaches. In still other embodiments, Veillonella and/or Prevotellabacteria is provided on the strip. As one of skill in the art willappreciate, the strip may be made to include at least one polymerselected from the group consisting of pullulan, hydroxypropylmethylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodiumalginate, polyethylene glycol, tragacanth gum, guar gum, acacia gum,arabic gum, polyacrylic acid, methylmethacrylate copolymer, carboxyvinylpolymer, amylose, high amylose starch, hydroxypropylated high amylosestarch, dextrin, pectin, chitin, chitosan, levan, elsinan, collagen,gelatin, zein, gluten, soy protein isolate, whey protein isolate, caseinand mixtures thereof. Preferably, the strip includes at least 0.2%xylitol by weight. In certain embodiments, the strip comprisesbioluminescent material. Preferably the strip is dissolvable in aperson's mouth within a period of 1 hour. In certain preferredembodiments, the strip has least one encapsulated feature that containsan agent selected from the group consisting of an antibiotic; lacticacid bacteria; and xylitol, e.g. at least 200 mg of xylitol. Such afrangible capsule may be constructed so that it may be broken by theindividual pressing against said strip with the individual's tongue.

Still other embodiments of the present invention are directed toaddressing the incidence of migraines and cluster headaches usingbacteria of the oral cavity and gut of an individual. Microbes, amongothers in the digestive tract, are capable of oxidizing alcohol toacetaldehyde. Compositions containing one or more cysteines as activeagents have been shown to bind acetaldehyde. These active agents havebeen found to also be capable of breaking down biofilms formed by somemicrobes, particularly in the stomach. One aspect of the presentinvention is therefore directed to reducing the likelihood of headaches,including not only migraines, but also cluster headaches. Thus, severalembodiments employ microbes, preferably particular bacterial species asset forth herein, to assist in decreasing the amount of acetaldehyde inthe body of an individual who suffers from such headaches. This can beachieved by employing cysteine generating microbes that are effective inreducing the amount of acetaldehydes. Other microbes are also effectivein degrading acetaldehydes.

While migraines are diagnosed more often in women, cluster headaches aremore prevalent in men. Cluster headache is a neurological disease thatinvolves, as its most prominent feature, excruciating unilateralheadaches of extreme intensity. “Cluster” refers to the tendency ofthese headaches to occur periodically, with active periods interruptedby spontaneous remissions. The cause of the disease is currentlyunknown. Cluster headaches are sometimes classified as vascularheadaches. The intense pain has been suggested to be linked with thedilation of blood vessels which creates pressure on the trigeminalnerve. Cluster headache episodes are known to be triggered by factors,such as alcohol consumption. Patients who are sensitive to alcohol notethat attacks are triggered within 5 to 45 minutes after the ingestion ofmodest amounts of alcohol, usually being less than a single cocktail orglass of wine. Alcohol triggers attacks in 70 to 80% of exposures.Microbial metabolism may contribute to the toxicity of alcohol,especially in the gastrointestinal tract, where aerobic and facultativeanaerobic bacteria convert ethanol to acetaldehyde. Indeed, acetaldehydeis known to be a highly toxic and pro-carcinogenic compound with variousnegative effects, ranging from DNA damage and impaired DNA excisionrepair to the degradation of folate. Thus one aspect of variousembodiments of the present invention is directed to providing particularbacteria to a person who consumes alcohol in a manner that such bacteriamay ameliorate the accumulation of acetaldehyde. In certain embodiments,bacteria are employed to degrade acetaldehyde, such bacteria preferablyhaving an aldehyde dehydrogenase, such as bacteria of the genusSaccharomyces and/or a threonine aldolase derived from Escherichia coli.Employment of CRISPR-Cas systems to incorporate the genes of suchbacteria into other bacteria as set forth herein forms various otherembodiments of the present invention. Thus, use of bacteria in the oralcavity, as well as in the gut microbiome of an individual, are employedto degrade acetaldehyde and thus reduce the incidence of migraines andcluster headaches. While many of the embodiments described herein areprincipally directed to a method and system for addressing the terriblepain occasioned by headaches caused by migraines and cluster headaches,one may further appreciate other aspects of the invention as beingdirected to hangover remedies as the ability to degrade alcohol andacetaldehyde to remedy the effects of consuming too much alcohol: thusintroducing “Hangover Strips” in addition to Headache Strips asdescribed herein.

One will appreciate that this Summary of the Invention is not intendedto be all encompassing and that the scope of the invention nor itsvarious embodiments, let alone the most important ones, are necessarilyencompassed by the above description. One of skill in the art willappreciate that the entire disclosure, as well as the incorporatedreferences, figures, etc. will provide a basis for the scope of thepresent invention as it may be claimed now and in future applications.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an illustration of a person experiencing the pain of amigraine headache.

FIG. 2(a)-(d) illustrate some exemplary surface architectural patternsaccording to the invention.

FIG. 3 is an illustration of how beneficial bacteria in an individual'sgut microbiome relates to the health of the central nervous system, aswell as how dysbiosis of the gut microbiome relates to various problemsexperienced with the central nervous system including the occurrences ofmigraine headaches.

FIG. 4 is an illustration of a pre-made sheet of strips that can bedisassociated with the sheet and then applied to mucosal membranes.

FIG. 5 is a side view of one embodiment of a strip having an outerlayer, an adhesive layer, a layer with an encapsulated agent containedthere between.

FIG. 6 is a side view of one embodiment where the encapsulated agent isencapsulated into small beads that are frangible via pressure of anindividual's tongue pressing against the strip so as to force it intothe roof of a person's mouth.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION

There is a higher prevalence of headaches among people who regularlyexperience GI symptoms compared to control groups without GI complaints.Colic has also been suggested as an early life expression of migraineand thus, the present inventors believe that there is an associationbetween migraine and infantile colic. Infantile colic is a common causeof inconsolable crying during the first months of life with incidencerates ranging from 5 to 19%. Mothers with a history of migraine were 2.6times as likely to have colic as infants than those mothers without amaternal history of migraine. Infants with abdominal colic have a lowerintestinal microbiota diversity and stability as compared to controlinfants in the first weeks of life. Children with migraines are morelikely to have experienced infantile colic compared with controls.

Irritable bowel syndrome (IBS) is a functional bowel disordercharacterized by abdominal pain, bloating, discomfort, and markedchanges in bowel habits. IBS and migraine are both 2-3 times moreprevalent in women than in men. IBS has been shown to be a disorder withan increased intestinal permeability and this permeability increaseswith more severe IBS symptoms.

The present inventors contend that there is a strong relationshipbetween GI disorders and migraine. One of the links between inflammatorydiseases and migraine are enhanced pro-inflammatory immune responses. Astrong trigger of pro-inflammatory immune responses is the leakage oflipopolysaccharides (LPS) from the intestinal lumen into thecirculation. Enhanced levels of LPS can enter the circulation when theintestinal permeability is increased. Depending on geneticsusceptibility, pro-inflammatory responses can occur in different partsof the body, e.g., in case of migraine on the nociceptors of thetrigeminal nerve.

Gut permeability and inflammation are bi-directionally related;increased permeability can cause inflammation, but inflammation can alsocause increased gut permeability. An increased gut permeability, andthereby increased translocation of LPS, can be caused by multiplefactors like medicines, exercise, mast cell activation, high fat diet,stress, etc. The most used method to measure epithelial barrier functionis with the lactulose/mannitol test. Mannitol is transported via thetranscellular pathway whereas lactulose is absorbed through theparacellular pathway. In case of increased permeability, more lactulosepasses the barrier and eventually ends up in the urine. Therefore, anincrease in intestinal permeability is characterized by an increasedratio of lactulose/mannitol. It is believed that the reduction of thepermeability of the intestine results in relief of migraine in thesubgroup of patients in whom intestinal permeability plays a role in thedisease. Subjects with food allergies have an increased intestinalpermeability compared with healthy controls.

The brain-gut-microbiome axis is the bidirectional communication betweenthe central nervous system and the gastrointestinal tract. Theunderlying mechanisms include increased gut permeability andinflammation. Probiotics decrease intestinal permeability as well asinflammation, and therefore reduce the frequency and/or intensity ofmigraine attacks. In accordance with various embodiments of the presentinvention, a combination of various bacterial strains may be employed tocombat migraines and dizziness including: Lactobacillus acidophilusDDS-1, Lactobacillus bulgaricus, Enterococcus faecium andBifidobacterium bifidum.

Probiotics are living microorganisms that have beneficial effects on thehealth of the host. The most used probiotics are lactobacilli andbifidobacteria. Effects of probiotics are dependent on the used speciesand strain. Probiotics in the treatment of GI disorders is believedeffective due to the strengthening of the intestinal barrier of aperson. Probiotics have shown to be able to improve the epithelialbarrier function via different mechanisms. Similarly, probiotics play arole in maintaining or improving gut barrier function as well as inmigraine patients with an enhanced intestinal permeability. Migraineprevalence is associated with gastrointestinal disorders. A combinationof different probiotics (Lactobacillus acidophilus, Lactobacillusbulgaricus, Enterococcus faecium, and Bifidobacterium bifidum) can beemployed for such purpose. Certain methods of the present invention aredirected to probiotic formulations included on or encapsulated into astrip of the present invention such that the desired bacteria isdelivered to the mucosal membrane. Such strips may include one or moredesired bacterial species, useful for promoting or maintaining thehealth and general well-being of humans, including but not limited tothe following: organisms of Enterococcus faecium, including strain NCIMB40371, deposited on 25 Feb. 1991 in accordance with the provisions ofthe Budapest Treaty, in the National Collections of Industrial andMarine Bacteria Limited, Aberdeen, under Accession No. NCIMB 40371.

Dental plaque, a sticky colorless film, is caused by bacterial depositsaccumulating on tooth or implant surfaces along the gingival margins andresults in the destruction of tooth-supporting tissues. Dental plaqueformation starts in cracks, grooves and surface roughness on teethand/or dental implants. In any given plaque sample, it is not uncommonto detect 30 or more bacterial species. Biofilms that colonizes thetooth surface may be among the most complex biofilms that exist innature. The bacteria associated with periodontal diseases reside inbiofilms both above and below the gingival margin. The supragingivalbiofilm is attached to the tooth or the implant and predominated byActinomyces species. The subgingival biofilm is typically more complexand can either attach to the tooth or implant, or to the gingivaltissue. Three microbe species are believed to be main players in thecause the periodontal diseases: A. actinomycetemcomitans, P. gingivalisand B. forsythus. Also, F. nucleatum, Campylobacter rectus, P.Intermedia, P. nigrescens, Eubacterium nodatum, P. micros and variousspirochetes have been singled out that may also be important species inperiodontal disease.

The destruction of tooth-supporting tissues results in a deepening ofthe space (periodontal pocket) between the root of the tooth and the gumtissue. Second to tooth decay, periodontal diseases are the mostfrequent oral diseases and may lead to partial or complete tooth or boneloss. It has been estimated that they affect as much as between 70-90%of the world population, and they are the major cause of tooth loss inpeople over 35 years of age. In periodontitis the infection hasprogressed to involve the oral tissues which retain the teeth in thejawbone. If untreated, periodontitis ultimately leads to loss of theaffected tooth. Chronic periodontitis, the most frequently occurringform of periodontitis, results in inflammation within the supportingtissues of the teeth, progressive loss of attachment as well asprogressive alveolar bone resorption. This form of periodontitis ischaracterized by pocket formation and/or recession of the gingiva. Asthe destruction advances, the mobility and movement of teeth increase,finally causing spontaneous loss of a tooth or a necessity of toothextraction. Treatment of periodontal diseases usually involves theremoval of bacterial deposits and dental calculus. However, it isdifficult to have full access for treating deeper periodontal pockets,resulting in remaining bacteria that may re-infect the tissue.

O₂ and NO act as environmental cues that trigger the coordinatedexpression of virulence genes and metabolic adaptations necessary forsurvival within a host. NO concentrations may be produced by fecalmicrobiota from nitrate, with the nitrate being reduced to ammonium bythe dissimilatory nitrate reduction to ammonium (DNRA) pathway.Gastrointestinal microbiota can generate substantial amounts of NO byDNRA, rather than by the generally accepted denitrification orL-arginine pathway. Bacterial nitrate reduction to ammonia, as well asthe related NO formation in the gut, is believed to be an importantaspect of the overall mammalian nitrate/nitrite/NO metabolism,demonstrating how the microbiome links diet and health.

Biofilm initiated diseases are by no means unique to the oral cavity.Approximately 65% of infections that affect the human are caused byorganisms growing in biofilms. These include dental caries, periodontaldisease, otitis media, musculoskeletal infections, necrotizingfasciitis, biliary tract infection, osteomyelitis, bacterialprostatitis, native valve endocarditis, meloidosis, prosthetic as wellas orthopedic complications and cystic fibrosis pneumonia.Characteristics are persistence and chronicity of the infections as wellas the difficulty in eradication.

The present inventors also submit that migraine headache is a precursorto Alzheimer's disease as it is associated with the same causativefactor, namely periodontitis. As such, and as addressed in the presentdescription and in co-pending cases, incorporated herein by thisreference, the treatment of migraines by addressing the breakdown ofbiofilms so as to kill particular bacteria, including spirochetes foundin the oral cavity. Strips of the present invention can be employed toposition agents, including beneficial bacteria, bacteria modifiedemploying CRISPR-Cas systems, and the use of antibiotics effectiveagainst various gram negative bacteria, including but not limited tospirochetes, forming various embodiments of the present invention. Thereare several aspects that are similar, if not the same in the processesof both illnesses. Risk factors common for patients suffering from eachdisorder include visual dysfunction, depression, lethargy, mood changes,sense of pain to non-painful stimuli, inability to produce purposefulcoordinated movements, and in women changes in ovarian hormone levels.The neurotransmitter systems and inflammatory processes throughout thebody are directly related to variations in estrogen and progesteronelevels.

One aspect of the present invention relates to the removal from the oralcavity of disease causing bacteria, principally gram negative bacteriaassociated with periodontitis, followed by antibiotic treatments toensure such bacteria removal from the oral cavity, and then followed upwithin hours with a regimen that includes the purposeful exposure of aperson's oral cavity with beneficial bacteria, thus promoting theavoidance of future periodontal disease. The strips as described hereincan be used for each or a combination of such functions. The correctformation of a beneficial biofilm is thus one aspect of the presentinvention. If this last step is not implemented, then there willinvariably be a biofilm generated, but often one that is not beneficialto the person, and one that could lead again to periodontitis. Thus, thepurposeful exposure and administration of select bacterial species isone objective of the present invention.

Streptococci constitute 60 to 90% of the bacteria that colonize theteeth in the first 4 hours after professional cleaning. Other earlycolonizers include Actinomyces spp., Capnocytophaga spp., Eikenellaspp., Haemophilus spp., Prevotella spp., Propionibacterium spp., andVeillonella spp.

Antibiotics can be prescribed at a low dose for longer term use, or as ashort-term medication to deter bacteria from re-colonizing. Preferably,in various embodiments of the present invention, strips that containappropriate amounts and types of antibiotics are employed to adjust thepopulation of the oral and the gut microbiome of a person to alleviatemigraine and dizziness symptoms. Incorporated by reference in itsentirety is U.S. Pat. No. 9,445,936, directed to the use of mucosalstrips, and especially oral strips, that can be provided with variousbacterial components to adjust and modify the oral microbiome of anindividual. It is sometimes advisable to undergo a treatment withantibiotics so as to reduce the number of other undesired bacteria inthe oral cavity, prior to the use of the strips having the desiredbacteria included thereon. Antibiotics which include tetracyclinehydrochloride, doxycycline, and minocycline are the primary drugs usedin periodontal treatment and that are preferred for use in the strips asdescribed herein. Such strips with these agents have antibacterialproperties, reduce inflammation and block collagenase (a protein whichdestroys the connective tissue). Metronidazole is generally used incombination with amoxicillin or tetracycline to combat inflammation andbacterial growth in severe or chronic periodontitis and the use of theseantibiotics on or encapsulated on strips of the present invention permitselective administration to the oral cavity in a manner that has neverbeen done before. In preferred embodiments, the direct delivery ofantibiotics to the surfaces of the gums by using the strips as describedherein is preferred and are extremely effective when used after scalingand root planing procedures. Among the various existing agents that canbe incorporated into the strips of the present invention, especiallythose that encapsulate such agents such that a person canself-administer the agents via tongue pressure applied to frangibleshells containing such material, are as follows: a doxycycline gel thatconforms to the contours of gum surfaces and solidifies over them;Chlorhexidine, a powerful antibacterial antiseptic; tetracyclinehydrochloride; metronidazole; and Minocycline. In certain embodiments,the strips as described herein are employed to modify the oralmicrobiome of an individual to treat migraines. Thus, a buccalbioadhesive strip is preferably used that has a first and second side,with the first side having a surface comprising a pattern defined by aplurality of spaced apart features each having at least one microscaledimension and having at least one neighboring feature having asubstantially different geometry, and wherein an average spacing betweenadjacent ones of said features is between 0.5 and 5 .mu.m in at least aportion of the surface. The first side has a bioadhesive that is adaptedto bind to a mucosal membrane for at least 1 hour while inside aperson's mouth. The strip may, for example, extend over a majority ofthe soft palate and preferably includes xylitol. Other embodiments havethe strip including at least one encapsulated pocket containing one ofan analgesic, a lactic acid bacteria, or another of the desired bacteriaas described herein. FIG. 4 is an illustration of a pre-made sheet 100of strips 120 that can be disassociated with the sheet and then appliedto mucosal membranes. FIG. 5 is a side view of one embodiment of a strip120 having an outer layer, an adhesive layer, a layer with anencapsulated agent contained there between. FIG. 6 is a side view of oneembodiment where the encapsulated agent 110 is encapsulated into smallbeads that are frangible via pressure of an individual's tongue pressingagainst the strip so as to force it into the roof of a person's mouth.

A major problem with bacterial biofilms is the inability of the hostimmune system and/or antibiotics and other antimicrobials to gain accessto the bacteria protected within the biofilm. Thus, a need exists tobreak through the protective barrier of biofilms to treat or kill theassociated bacterial infections as the biofilm can act as a reservoirfor future acute infections often with lethal consequences. Although alaser can be used to kill bacteria, this method in isolation does notnecessarily remove the bacteria, and thus a biofilm can remain on theimplant which can hinder osseointegration and may act as a source oflater infection. Antimicrobial agents are not effective at normaldosage, as the minimum inhibitory concentration for antibiotics for anorganism in biofilm mode might be 1000-1500 times higher than for thesame organism in the planktonic state. Periodontal diseases areinfections caused by microorganisms that colonize the tooth or implantsurface at or below the gingival margin. While these infections havemany properties in common with other infectious diseases, they exhibitunique properties conferred by their site of colonization and the natureof the environment in which they reside. The onset of the diseases isusually delayed for prolonged periods of time after initial colonizationby the pathogens.

Thus certain embodiments of the present invention are directed to aprophylactic method for treating chronic migraine comprising (after theabove referenced steps of removing pathogenic bacteria associated with aperson's periodontitis, including after antibiotic applications)administering on a daily basis to humans a composition comprising:probiotics selected from the group consisting DDS-1 strain ofLactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillusjohnsonii, Bifidobacterium bifidum and Enterococcus faecium in a unitdosage amount ranging from 1000 to 3000 mg. Useful probiotic agentsinclude (in addition to the numerous others referenced herein)Bifidobacterium sp. or Lactobacillus species or strains, e.g., L.acidophilus, L. rhamnosus, L. plantarum, L. reuteri, L. paracasei subsp.paracasei, or L. casei Shirota; Lachnospira, Veillonella,Faecalibacterium and Rothia, and Prevotella.

Human oral bacteria interact with their environment by attaching tosurfaces and establishing mixed-species communities. Several orbacterial species are amenable to genetic manipulation for molecularcharacterization of communication both among bacteria and betweenbacteria and the host. Due to the dynamics of growth and adherence, thebacterial populations in the oral cavity are constantly changing, evenduring the intervals between normal daily oral hygiene treatments. Thevarious species within oral biofilms function as a coordinated communitythat uses intra- and interspecies communication.

Development of the oral microbial community involves competition as wellas cooperation among the 500 species that compose this community,including the following: Actinobacillus actinomycetemcomitans,Actinomyces israelii, Actinomyces naeslundii, Capnocytophaga gingivalis,Capnocytophaga ochracea, Capnocytophaga sputigena, Eikenella corrodens,Eubacterium spp., Fusobacterium nucleatum, Haemophilus parainfluenzae,Porphyromonas gingivalis, Prevotella denticola, Prevotella intermedia,Prevotella loescheii, Propionibacterium acnes, Selenomonas flueggei,Streptococcus gordonii, Streptococcus mitis, Streptococcus oralis,Streptococcus sanguis, Treponema spp., and Veillonella atypica. AlthoughF. nucleatum is often considered a periodontal pathogen, it may insteadcontribute to maintaining homeostasis and improving host defense againsttrue pathogens.

Competitive and cooperative mechanisms may be central to successfulmixed species colonization as well as the proper succession of generaknown to occur on teeth in both health and disease. Within the oralcavity, bacteria form multispecies communities that are distinguishableprimarily by their location (supragingival versus subgingival versusepithelial). The subgingival community has the highest species richnessand the greatest capacity for pathogenic outcome, such as periodontaltissue destruction.

It has been shown in vivo that veillonellae are not capable ofcolonizing the tooth surface without streptococci as metabolic partnersand that larger populations of veillonellae develop in coculture withstreptococci that recognize them as a coaggregation partner than incoculture with streptococci with which they do not coaggregate.

While migraines and Alzheimer's Disease share certain similarities interms of bacterial and nerve passages of material to the brain, it is asyet unknown if the spirochetes believed responsible for the majority ofAD is also associated with migraines and cluster headaches. About 60oral species of Treponema have been identified, and spirochetesconstitute a large percentage of the total oral bacterial numbers.Accordingly, a large T. denticola population could benefit greatlythrough interaction with a small P. gingivalis population. A stimulatoryeffect of P. gingivalis supernatant on T. denticola growth points to asynergistic interaction between P. gingivalis and other anaerobicbacteria such as oral spirochetes, and may be increased growth andincreased virulence of these potential periodontal pathogens.

One aspect of the present invention is to avoid a large population ofspirochetes in the oral cavity and one way of doing so involves limitingthe population of other bacteria that spirochetes depend upon to thriveand grow. Thus, one aspect of the present invention is directed to thelimitation or destruction or the retarding of growth conditions forspirochetes, which includes the limitation of the presence of P.gingivalis. Subtle regulation of gene expression in any organism withina community may lead to significant changes in the organism's ability toparticipate in community activities, such as use of community-formedmetabolic end products as nutrients. Thus, interference with thebiofilms relied upon for spirochetes growth and maintenance is oneaspect of the present invention, which may be achieved via the use oforal strips of the present invention that have structural featuresthereon that deter bacteria growth, and that may also have antibioticsas well as beneficial bacteria residing thereon, and alternatively or inaddition to, may have xylitol on the strip.

One aspect of the present invention is to trigger small changes in aperson's oral and/or gut microbiome such that they cause large shifts inpopulation composition and metabolic output of mixed-speciescommunities. In certain embodiments, this is accomplished byinactivation of a gene (via CRISPR-Cas or CRISPR-Cpf1) involved inmixed-species community formation to cause a subtle variation in anorganism's phenotype only during critical transitions in populationcomposition and have no effect on population composition before or afterthe transition.

In still further particular embodiments, the Treponema denticola genomeis modified to target the expression of particular chromosomalintegrons, as it is the only human-associated bacterial species thatharbors chromosomal integrons, with no integrons in other Treponemaspecies being found. For example, in one particular embodiment of thepresent invention, genes from Treponema are modified to excise one ormore virulence factors to address the progression of diseases, such asAlzheimer's disease, dizziness, migraines and cluster headaches, byaddressing the causative factors of such diseases in the oral cavity ofthe person, prior to the full-blown development of such diseases.Chronic periodontitis is an inflammatory disease that is caused by theaccumulation of bacteria in the form of a biofilm in the periodontalpocket. It can be treated with oral hygiene in conjunction with β-lactamantibiotics. Many oral anaerobic bacteria associated with chronicperiodontal diseases have developed resistance to β-lactam antibioticsby virtue of their production of β-lactamase enzymes. Using CRISPR-Castechniques to delete virulence factors and to restore antibioticsensitivity to permit use of known effective antibiotics, is one aspectof the present invention.

A high prevalence of β-lactamase-producing oral anaerobic bacteria hasbeen found in patients with chronic periodontitis. As a large percentageof bacteria carry a gene that renders them resistant to β-lactamantibiotics, alternative antimicrobial agents should be employed inpatients that are non-responsive to β-lactam antibiotics. Use ofCRISPR-Cas systems to render particular bacteria susceptible to suchantibiotics is one aspect of the present invention, as well as thepurposeful exposure of a person's oral cavity (after existing bacterialflora has been substantially removed) to replace the flora with aCRISPR-Cas system modified bacteria culture such that the control overthe oral population of bacteria can be achieved, such as by renderingsuch bacteria susceptible to antibiotics.

Yet another aspect of the present set invention is directed to theemployment of phospholipid vesicles in addressing desired modificationsto the human microbiome, and in particular to the oral microbiome.Bacterial membrane vesicles (MVs), released by many bacteria, mainlyconsist of the cell membrane and typically range from 20 to 400 nm insize. Bacterial MVs are involved in several biological functions, suchas delivery of cargo, virulence and gene transfer. Although MVbiogenesis and biological roles are yet to be fully understood, oneaspect of the present invention relates to the genetic engineering ofsuch MVs to tailor them for applications in drug delivery systems andnanobiocatalysts, MV vaccines, etc. MVs have been found to mediatediverse functions, including promoting pathogenesis, enabling bacterialsurvival during stress conditions and regulating microbial interactionswithin bacterial communities. Modification and increased expression ofsuch vesicles, including the ability to employ CRISPR-Cas systems toaffect the transfer of desired components to the oral cavity via suchvesicles, is part of various embodiments of the present invention. Theexistence of membrane vesicles increases the complexity involved in thediffusion of secreted substances during microbial interactions and MVsecretion has been observed in Gram-negative bacteria as well as inother prokaryotes, including Gram-positive bacteria and archaea. MVscontain proteins, DNA, RNA and quorum sensing signals, and thesesubstances are transferred to cells. MVs have unique characteristics,including the fact that several chemical substances are highlyconcentrated in MVs, the interior substances in MVs are protectedagainst environmental stresses, and MVs play a role in effectivelydelivering these substances to cells.

In one particular aspect of the present invention, MVs of human specificpathogens are employed to incapacitate the pathogenic nature of suchbacteria. The association between MVs and eukaryotic cells has beenstudied in pathogenic bacteria, and MVs secreted from pathogens transfervirulent factors to cells. In particular, specific proteins localized onthe surface of MVs increase the association with epithelial cells,believed to be due to increasing the association of MVlipopolysaccharide with cells.

Microbial predation using MVs occurs when virulent factors orpeptidoglycan hydrolytic enzymes contained in MVs are transferred toother bacterial cells. It has been suggested that the mechanism ofbacterial lysis via MVs secreted from Gram-negative bacteria differs inwhether recipient cells are Gram-negative or positive. Thus,cell-to-cell communications in the oral cavity involve microbesintricately communicating through methods using MVs, thereby influencinginterspecies networks, microbial community organization and ecosystemdynamics. Employment of the specially surfaced structured strips of thepresent invention may be used to alter the population of an individual'smicrobiome in a manner that can later the progression of bacterialrelated diseases, including migraines, cluster headaches andAlzheimer's. With respect to treatment, several embodiments employGraphene oxide nanosheets, and especially in the form of the strips asdescribed herein, to deliver an effective antibacterial material againstdental pathogens, including especially Treponema denticola.

As discussed herein, there is an association with periodontitis andmigraines. Thus, one aspect of certain embodiments of the presentinvention relate to a series of steps to be undertaken to address thekilling and elimination of certain gram negative bacteria that areassociated with periodontitis, followed by the purposeful application ofa composition having beneficial bacteria that are adapted to growth soas to populate a person's mouth and thus prevent the reestablishment ofharmful bacteria that could, if permitted to persist in a person'smouth, lead to various maladies, including periodontitis, Alzheimer'sdisease, cluster headaches and migraines. In particular embodiments,strips are employed that have at least one encapsulated drug containingcapsule that when broken or fractured, can release a predeterminedamount of a drug, such as one effective to reduce if not eliminatecertain gram negative bacteria and/or spirochetes that are believedresponsible for periodontal disease, and in one preferred embodimentinvolves the use of metronidazole, preferably encapsulated or imbuedonto a bioadhesive strip of the present invention. Certain embodimentsof the present invention employ oral pharmaceutical compositions thatinclude metronidazole, especially contained within a release layer of abioadhesive strip that dissolves or erodes in the oral cavity.

Thus, in certain embodiments, the present invention provides an abilityof a patient to purposefully cause the rupture of an encapsulated packetor pocket (e.g. a space in a strip that captures the agent of choice,which is released upon the rupture of such packet/pocket) that isassociated with a strip that is adapted to be placed in association witha person's gums, with the encapsulated material preferably being anantibiotic, e.g. metronidazole, adapted to kill gram negative bacteria,and especially microbes associated as a causative agent inperiodontitis.

Metronidazole is a nitroimidazole antibiotic with antibacterial activityagainst obligate anaerobic bacteria and certain protozoan parasites. Theusual oral antibacterial therapies for treating pathologies have oftengiven contradictory results. For instance, excessive dilution of theactive ingredient has been observed in the intestinal lumen. Thisdilution is believed to be due to the premature release of theantibacterial agent from the pharmaceutical form even before reachingthe duodenum such as in the stomach and in the immediate vicinity of thepatient's pyloric valve. Similarly, in the oral cavity, use ofmetronidazole in a systemic fashion has limited results, as it does notpersist in a concentrated enough form to kill undesired microbes thatare entrenched in the gum regions of a person's mouth. Thus, in variousembodiments of the present invention, metronidazole is provided inadhesive strips that are configured for providing the oral cavity with asufficiently high level and dosage of the drug to accomplish the desiredkilling of certain bacterial species, including particular spirochetessuch as T. denticola.

In another particular aspect, a genetically modified microbe, such as abacteria of the species T. denticola and/or Prevotella, includes aninducible promoter directing expression of an essential protein and/oris modified such that expression of virulence factors are substantiallyreduced or eliminated. In certain embodiments, a composition comprisesone or more genetically modified microbes, such as a bacteria of thespecies T. denticola and/or Prevotella, each of which are geneticallymodified, and especially by employment of CRISPR-Cas or Cpf1 systems toattenuate virulence factors, etc. CRISPR-Cas or Cpf1 modified microbesin which expression of an endogenous pathogenic protein is substantiallyreduced or eliminated in the one or more genetically modified bacteriainclude an inducible promoter regulating the expression of a virulencefactor for such microbe.

Still other embodiments are directed to the use of bacteriophagesmodified to attack particular bacteria, especially T. denticola and/orPrevotella, to reduce the populations of one or the other in the oralcavity. In accordance with the present invention, native bacterialadaptive immune systems can be modified to thwart the conventionalability to confer immunity against bacteriophage infection. TheCRISPR-Cas sequences, which are present in approximately 40% ofeubacterial genomes and nearly all archaeal genomes sequenced to date,is employed to reverse the resistance to various antimicrobial agentssuch as small molecule antibiotics and bacteriophages.

Thus in certain embodiments, the innovative method is directed todecreasing the relative representation of a specific strain of bacteria,preferably T. denticola and/or Prevotella, within a heterogenouspopulation of oral bacteria, comprising contacting the heterogenouspopulation of oral bacteria with a bacteriophage comprising apolynucleotide that expresses (a) an RNA-directed DNA-bindingpolypeptide comprising a nuclease module; and (b) a targeting modulecomprising a guide RNA, wherein the targeting module tethers theRNA-directed DNA-binding polypeptide to a target DNA sequence within,thereby producing a double-strand break within the target sequence,wherein the target sequence is unique to the specific strain of T.denticola and/or Prevotella bacteria.

In certain embodiments of the present invention, delivery of beneficialbacteria, after the removal of pathogenic bacteria and also after use ofthe strip treatments as described herein (e.g. including theadministration of local antibiotics to oral tissues), is achieved in amanner that comports with where such bacteria are normally located in aperson's body. For example, many of the bacteria that confer protectionagainst autoimmune diseases and that are otherwise believed to promotehealth in humans, and as described herein, are normally resident in thehuman mouth. For instance, one of the FLVR bacteria recently touted asbeing beneficial in the prevention of disease, namely Veillonella, iscommonly found in a person's mouth, mostly living on the tongue andsaliva. In various embodiments, selected bacteria, such as Veillonella,is purposefully presented on an oral strip that adheres to the mucosalmembrane of a person.

In various embodiments of the present invention, bacterial species to beexposed to a person's oral (or in other embodiments, gut) microbiome,include those specifically modified by employing the CRISPR-Cas andCRISPR-Cpf1 systems to render the virulence factors of various bacteriaineffective. CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats) is a prokaryotic adaptive defense system that providesresistance against alien replicons such as viruses and plasmids. CRISPRsevolved in bacteria as an adaptive immune system to defend against viralattack. Upon exposure to a virus, short segments of viral DNA areintegrated into the CRISPR locus. RNA is transcribed from a portion ofthe CRISPR locus that includes the viral sequence. In certain preferredembodiments, rather than using CRISPR-Cas, one employs theCRISPR-associated endonuclease Cpf1. e.g. a CRISPR from Prevotella andFrancisella 1 (Cpf1) nuclease for CRISPR-based genome editing, andincorporating 20150252358 to Maeder by this reference).

CRISPR-Cpf1 is a class II CRISPR effector that is distinct from Cas9,and is a single RNA-guided endonuclease that uses T-rich PAMs andgenerates staggered DNA double stranded breaks instead of blunt ends.Its smaller protein size and single RNA guide requirement makes CRISPRapplications simpler and with more precise control. The human gut is arich habitat populated by numerous microorganisms, each having a CRISPRsystem. To comply with written description and enablement requirements,incorporated herein by the following references are the following patentpublications: 2014/0349405 to Sontheimer; 2014/0377278 to Elinav;2014/0045744 to Gordon; 2013/0259834 to Klaenhammer; 2013/0157876 toLynch; 2012/0276143 to O'Mahony; 2015/0064138 to Lu; 2009/0205083 toGupta et al.; 2015/0132263 to Liu; and 2014/0068797 to Doudna;2014/0255351 to Berstad et al.; 2015/0086581 to Li; 20160311913 to Sun;PCT/US2014/036849 and WO 2013/026000 to BRYAN.

In various aspects of the present invention, CRISPR is employed tomodify aspects for both bacterial and helminthes gene expression suchthat undesired normally transcribed proteins are excised or precludedfrom being expressed, thus precluding the deleterious effects of suchproteins. Thus, normally dangerous species of bacteria and helminthes(from a perspective of such bacteria or helminthes causing disease in ahuman) can be modified so that such undesired effects of bacterial andhelminthes infection are disrupted or deleted or lessened in a fashionthat still permits the beneficial aspects of bacterial and helminthesproteins to be maintained.

Various embodiments of the present invention combine each of the abovereferenced four FLVR bacteria and using CRIPR, pathogenic and/or toxicelements are excised to preclude detrimental health issues that wouldnormally be encountered using one or more of such bacteria, whilepreserving the immune system attributes attained by the presence of suchbacteria. Preferably, the CRISPR modified bacteria of the FLVR speciesare then combined in a formulation suitable for use as either an oralcomposition (preferably administered via the strips as describedherein). In certain embodiments, treatment of newborns so as to promotebeneficial gut and oral microbiomes is achieved via strips or a vaginalcream (for the mother) such that a newborn is first exposed to suchbacteria when traveling down the birth canal.

In certain embodiments of the present invention, antibiotic resistanceof certain bacteria is modulated by employment of CRISPR to insert intothe genome of a bacteria antibacterial sensitivity such that it canselectively be killed, if necessary, after it is employed to triggerdesired immune responses in a new born or other individual. Thus, thevarious bacterial and helminthes species mentioned herein that areincluded in an or added to an Amish-soil may, in certain embodiments, beextracted and modified using CRISPR methods to do one of several things,including adding antibiotic sensitivity to various species so that theycan be employed for triggering immune responses of an individual, andthen later killed or rendered ineffective by the use of targetedantibiotics or anti-helminthes drugs.

In particular embodiments, directed to a topical composition of abacterial and/or helminthes containing composition, such compositionincludes cells that have been transformed by use of CRISPR to deleteparticular undesired attributes of wild-type species, including theexpression of disease causing proteins. Thus, creams, ointments, etc.may be employed as a vaginal pre-birth composition so that a new born,traveling down the vaginal canal, is exposed to a plurality ofbeneficial bacteria and/or helminthes proteins and other constituentssuch that a new born innate immune response is triggered to protect thenew born from developing the variety of allergic and autoimmune diseasesas described herein. In still other embodiments, a suitable oral ormucosal agent is administered to an individual's microbiome to provide away to alter the existing microbiome of the individual such thatmigraines and cluster headaches can be prevented.

The use of CRISPR to tailor bacterial and helminthes components toeither add desired characteristics and/or to delete known deleteriousaspects of such bacteria or helminthes, provides a novel system andmethod for treating a variety of diseases such that bacteria andhelminthes that would normally be considered too dangerous to employ asan agent to treat allergic, autoimmune conditions, migraines, clusterheadaches, etc. is now rendered available for such purposes.

In various embodiments, DNA is injected into bacteria to restoreantibiotic sensitivity to drug-resistant bacteria, and to also preventthe transfer of genes that create that resistance among bacteria. TheCRISPR-CAS system may also be employed to render certain bacteriasensitized to certain antibiotics such that specific chemical agents canselectively choose those bacteria more susceptible to antibiotics, see,e.g. US Pat. Publication No. 2013/0315869 to Qimron, which isincorporated in its entirety by this reference.

The microbiome of an individual is disrupted by antibiotics and thus,the employment of CRISPR as a way to bypass common modes of multidrugresistance, while being selective for individual strains, is employed invarious embodiments of the present invention to attain the benefitsderived by the presence of particular bacteria and helminthes, includingthe triggering of desired immune development by newborns and otherindividuals, (e.g. those with multiple sclerosis, etc.) as well as inaddressing the treatment and avoidance of migraines and clusterheadaches. CRISPR-Cas systems employ CRISPR RNAs to recognize anddestroy complementary nucleic acids. In various embodiments of thepresent invention, CRISPR-Cas systems are used as programmableantimicrobials to selectively kill bacterial species and strains suchthat desired selected targets can be focused on such that virtually anygenomic location may be a distinct target for CRISPR-basedantimicrobials, and that, in conjunction with an appropriate deliveryvehicle, such as those employed by Bikard et al. and Citorik et al., oneis able to effectively deploy a CRISPR-Cas system as an antimicrobialagent.

Another aspect of certain embodiments includes making syntheticCRISPR-containing RNAs that target genes of interest and using them withCas enzymes. The specificity of CRISPR-Cas systems permits one to designmethods to target a single bacterial species so that only essentialgenes from that one species is targeted and cut up. CRISPR-Cas systemsare employed in various ways in the many embodiments of the presentinvention to retain the beneficial bacterial communities intact and tooffer protection against undesired bacterial pathogens.

CRISPR has a certain protein in it called Cas9 that acts like a scissoras it recognizes specific sequences of DNA and cuts it enabling one toperform genome-editing of a bacterial genome in a person's microbiome.There exists another CRISPR system, CRISPR-Cpf1 that is even morepreferred for use in microbial systems. Cpf1 is important in bacterialimmunity and is well adapted to slice target DNAs. Cpf1 prefers a “TTN”PAM motif that is located 5′ to its protospacer target—not 3′, as perCas9, making it distinct in having a PAM that is not G-rich and is onthe opposite side of the protospacer. Cpf1 binds a crRNA that carriesthe protospacer sequence for base-pairing the target. Unlike Cas9, Cpf1does not require a separate tracrRNA and is devoid of a tracrRNA gene atthe Cpf1-CRISPR locus, which means that Cpf1 merely requires a cRNA thatis about 43 bases long—of which 24 nt is protospacer and 19 nt is theconstitutive direct repeat sequence. In contrast, the single RNA thatCas9 needs is ˜100 nt long.

The CRISPR system may be employed in various embodiments to strengthenantibiotics or to kill the bacteria altogether. By removing thebacteria's genes that make them antibiotic-resistant, CRISPR can boostthe effectiveness of existing drugs. CRISPR can also be used to remove abacteria's genes that make them deadly and facilitate RNA-guidedsite-specific DNA cleavage. Analogous to the search function in modemword processors, Cas9 can be guided to specific locations within complexgenomes by a short RNA search string.

In certain embodiments, various particular bacterial species are focusedon to delete or modulate their gene expressions, such species includingthe following: Streptococcus; Escherichia coli, Streptococcus pyogenes,and Staphylococcus epidermidis. This prokaryotic viral defense systemhas become one of the most powerful and versatile platforms forengineering biology.

In various embodiments, the CRISPR-Cas systems is employed to controlthe composition of the gut flora or oral microbiome, such as bycircumventing commonly transmitted modes of antibiotic resistance anddistinguishing between beneficial and pathogenic bacteria. Forapplications that require the removal of more than one strain, multiplespacers that target shared or unique sequences may be encoded in asingle CRISPR array and/or such arrays may be combined with a completeset of cas genes to instigate removal of strains lacking functionalCRISPR-Cas systems. Because of the sequence specificity of targeting,CRISPR-Cas systems may be used to distinguish strains separated by onlya few base pairs. The specificity of targeting with CRISPR RNAs may beemployed to readily distinguish between highly similar strains in pureor mixed cultures. Thus, in certain embodiments, varying the collectionof delivered CRISPR RNAs is employed to quantitatively control therelative number of individual strains within a mixed culture in a mannerto circumvent multidrug resistance and to differentiate betweenpathogenic and beneficial microorganisms.

In certain other aspects, particular embodiments of the presentinvention are directed to the use of CRISPR to excise certain priorinfectious adenovirus DNA sequences that are considered responsible forthe increased obesity of individuals harboring the same. Reference ismade to Kovarik, U.S. Pat. No. 8,585,588, “Method and system forpreventing virus-related obesity and obesity related diseases.” Afterdetermining whether one has been infected with a particular virus, theviral DNA can then be excised via CRISPR-Cas to remove the previouslyinserted DNA, thus effectively reducing if not eliminating theadenovirus gene from the individual. Thereafter, to avoid being infectedwith such adenovirus again, practice of the method as set forth in U.S.Pat. No. 8,585,588 will lessen, if not prevent, reacquisition of suchvirus.

Controlling the composition of microbial populations is important in thecontext of desiring to expose individuals to particular species ofbacterial and other microbes, helminthes, etc. and especially those thathave not been previously exposed to antibiotics, antimicrobial peptides,and lytic bacteriophages. Use of CRISPR-Cas provides a generalized andprogrammable strategy that can distinguish between closely relatedmicroorganisms and allows for fine control over the composition of amicrobial population for use in the present invention. Thus, the RNAdirected immune systems in bacteria and archaea called CRISPR-Cassystems is employed in various embodiments of the present invention toselectively and quantitatively remove and/or alter individual bacterialstrains based on sequence information to enable the fine tuning ofexposure of desired antigens. Thus, such genome targeting usingCRISPR-Cas systems allows one to specifically remove and/or alterindividual microbial species and strains in desired ways.

In various embodiments, it is desirable to remove—using CRISPR-Cassystems—particular viable genes in pathogenic bacteria and/or otherpathogenic portions (e.g plasmids, etc. of such bacteria)—while sparingother desired commensal bacteria, in order to provide exposure todesired immune developing proteins.

In various embodiments, one of skill in the art will appreciate thatremoval or alteration of particular strains of bacteria may be achievedusing both type I and type II CRISPR-Cas systems, given the distinctionbetween these systems being that type I systems cleave and degrade DNAthrough the action of a 3 ‘-to-5’ exonuclease, whereas type II systemsonly cleave DNA. In still other embodiments, multiple guide RNAs canalso be used to target several genes at once. The use of effectorfusions may also expand the variety of genome engineering modalitiesachievable using Cas9. For example, a variety of proteins or RNAs may betethered to Cas9 or sgRNA to alter transcription states of specificgenomic loci, monitor chromatin states, or even rearrange thethree-dimensional organization of the genome.

Because preferred embodiments relate to the modification ofmicrobes—rather than to the human genome—and especially only thosemicrobes that show tropism for humans, the unintended consequences ofemploying Crispr-Cas on organisms is lessened, if not eliminated.Moreover, use of CRISPR-Cas to also insert genes that have controllableelements such that the cells are killed by triggering the expression ofsuch genes, is another way to reduce if not eliminate concerns about anunintended release of a modified organism. These types of controls arewell known to those of skill in the art and have been long employed, forexample, by those involved in creating genetically engineered organisms,such as by inserting genes so that organisms become susceptible tovarious conditions, such as temperature, antibiotic exposure, etc., suchthat microbes that may somehow escape desired conditions will not beviable. Particular embodiments of the present invention are directed tothe employment of four specific bacterial genera—Lachnospira,Veillonella, Faecalibacterium and Rothia. Modifying the human genome,made possible by the CRIPSR technique, has its own wonderful upsides andequally daunting downsides. Permanent deletion of genes from the humangenome is much more controversial than deletion of bacterial genes.Thus, one desirable aspect of the present invention is directed to thefar less controversial modification of gut microbes resident in thehuman being to promote health and to trigger the desired immuneresponses as described herein.

Faecalibacterium prausnitzii, which represent more than 5% of thebacteria in the intestine, is encouraged to populate the guts ofpatients. Such enhanced growth of this bacterium may also be employed tocombat certain forms of inflammatory bowel disease. In variousembodiments of the present invention, Enterococcus faecalis is aresubjected to CRISPR-Cas procedures to remove undesired virulence andpathogenicity factors, such as several genes isolated from resistantenterococci (agg, gelE, ace, cy1LLS, esp, cpd, fsrB) which encodevirulence factors such as the production of gelatinase and hemolysin,adherence to caco-2 and hep-2 cells, and capacity for biofilm formation.Deletion and removal of certain antibiotic resistance, for example theacquisition of vancomycin resistance by enterococci, is desired also soas to properly and safely employ this bacteria in the present invention.In a particular embodiment, the addition of E. faecalis LAB3 1 isemployed to trigger desired immune system responses.

CRISPR-Cas can be used on the various identified microbiome constituentsto modify gene expression, including cutting of a gene, repress oractivate a gene, etc. It can be employed to deliver desired regulatorsor any protein to a desired place on a genome of a microbe, thuspermitting one to tailor the attributes of the microbiome of anindividual to promote the health thereof, including the programmedtriggering of particular immune responses in an infant. BecauseCRISPR-Cas acts before transcription occurs, it is able to be employedto target regulatory and other elements on the DNA of microbes that makeup the microbiome. In certain embodiments, CRISPR-Cas is employed todeliver fluorescent markers to certain DNA sequences, thus permittingone to determine whether any particular sample has been treated inaccordance with the present invention, thus ensuring, for example,identity of various materials, safety issues, types of enhanced soils,etc. This permits labeling of living cells with a desired color.

Many embodiments rely upon the ability to deliver agents via mucosaladhesive strips, such as described, for example, in U.S. Pat. No.8,701,671, which is fully incorporated herein by this reference. In sucha manner, one objective is to accept the beneficial traits of themicrobiome's interaction with the human immune system while avoiding theinfectious aspects of bacterial, viral and helminth aspects of suchexposure to a human being. Thus, in various embodiments of the presentinvention, the engineering of communal bacteria with improved propertiesusing a CRISPR/Cas system is employed. Thus, in certain embodiments thepresent invention is directed to delivering to microbial cells in vivo adelivery vehicle with at least one nucleic acid encoding a gene ornucleotide sequence of interest, such method employing an RNA-guidednuclease. The microbial cells may be either or both pathogenic microbialcells or non-pathogenic bacterial cells and the gene or nucleotidesequence of interest may be a virulence factor gene, a toxin gene, anantibiotic resistance gene, or a modulatory gene.

There exist various concerns about how CRISPR-Cas systems and methodwill be employed with respect to attempting to improve human healththrough and using a technology that cuts sections of DNA out of agenome, effecting permanent changes to the human DNA. Indeed, many inthe scientific community are considering whether a moratorium on the useof this powerful and yet simple technology should be implemented untilsuch time as all the risks involved can be better assessed. In thecontext of the present invention, however, this particular issue iseither absent or of lesser importance due to one focus of manyembodiments being relegated to the modification of DNA of the microbegenomes, rather than the human genome. Thus, the present invention isone way in which the human health concerns can be benefited directly bythe use of a DNA deletion system without affecting the long term andpermanent deletion of human genes. It is not believed to be obvious, letalone intuitive, that human health can be benefited by such a DNAdeletion system used in a fashion that affects only gut microbes in ahuman's system. Moreover, the use of such a DNA modification system formicrobes, but not for the direct deletion of genes from a human, and theuse of such a system prior to the exposure of a human to such modifiedmicrobes, has not previously been done, especially with the added stepof modifying select microbes having immune beneficial attributes—andespecially using modified microbes that one would otherwise haveconsidered to be pathogenic.

Individuals who have regular contact with livestock, such as farmers andtheir wives, have bacterial communities dominated by Prevotella, a typeof bacteria that is also abundant in the gut microbiota of cattle andsheep. Prevotella are among the most numerous microbes culturable fromthe rumen and hind gut of cattle and sheep. Percentages vary butPrevotella is often the most common bacterial genus in the cattle. Whilecertain aspects of particular embodiments are directed to the Prevotellagenus, others are more focused on particular species within such genus,namely P. intermedia. The present inventors contend that thecontributions of microbes to multiple aspects of human physiology andneurobiology in health and disease have up until now not been fullyappreciated.

Treponema denticola is an oral anaerobic spirochete closely associatedwith the pathogenesis of periodontal disease—and the present inventorsbelieve it is associated with numerous systemic diseases, includingAlzheimer's disease. The T. denticola major surface protein (MSP),involved in adhesion and cytotoxicity, and the dentilisin serineprotease are key virulence factors of this organism. Thus, one aspect ofthe present invention relates to the use of CRISPR-Cas or Cpf1 to targetthese virulence factors and thus, excise them from T. denticola so as torender it susceptible to antibiotics so as to reduce its presence in theoral microbiome, thus advancing the prevention of not only migraines,cluster headeaches and dizziness, but for Alzheimer's disease as well.Periodontal diseases are polybacterially induced, multifactorialinflammatory processes of the tooth attachment apparatus and are theprimary cause of tooth loss after the age of 35. The ability of suchdisease to escape detection and the failure of many to regularly visit adentist to diagnose such a disease, leads to the prevalence ofAlzheimer's disease as we see today. The elderly often show neglect oforal hygiene which can stimulate recurrent chronic oral infection, whichpromotes inflammation and then leads to confusion and dementia.Interfering with inflammation is thus one objective of the presentinvention and in certain embodiments, it is beneficial to combineanti-inflammatory agents with antibacterials.

The periodontopathogenic spirochete T. denticola possesses a number ofvirulence factors including motility, the ability to attach to hosttissues, coaggregation with other oral bacteria, complement evasionmechanisms, and the presence of several outer sheath and periplasmicproteolytic and peptidolytic activities. Two components associated withthe spirochetes' outer sheaths and extracellular vesicles are the majorsurface protein (also known as the major outer sheath protein [MSP]) anda serine protease, dentilisin, previously known as the chymotrypsin-likeprotease. Recent bioinformatics analysis reclassified dentilisin as amember of the subtilisin rather than the chymotrypsin family. Dentilisinis involved in the degradation of membrane basement proteins (laminin,fibronectin, and collagen IV), serum proteins (fibrinogen, transferrin,IgG, and IgA), including protease inhibitors (al-antitrypsin,antichymotrypsin, antithrombin, and antiplasmin), and bioactivepeptides. Degradation of tight junction proteins by dentilisin seems toenable the penetration of epithelial cell layers by this oralspirochete. MSP is a major antigen with pore-forming activity. Thisabundant membrane protein mediates the binding of T. denticola tofibronectin, fibrinogen, laminin, and collagen, induces macrophagetolerance to further activation with lipopolysaccharide (LPS), andelicits cytotoxic effects in different cell types.

One object of the present invention is to employ LL-37 against T.denticola, especially employing the strips as set forth herein. Salivainhibits dentilisin, attenuating its virulence properties but conservingLL-37 activity. Thus one aspect of the present invention is directed tothe use of LL-37 to kill T. denticola. The human host defense peptideLL-37 is preferably administered via the strips of the presentinvention, especially those having encapsulated pockets of the agentsuch that administration thereof can be achieved by the patient upontongue pressure being applied to a frangible shell present as part ofthe strip. Deficiency in the human host defense peptide LL-37 haspreviously been correlated with severe periodontal disease. Treponemadenticola is an oral anaerobic spirochete closely associated with thepathogenesis of periodontal disease. Treponema denticola is an importantperiodontal pathogen capable of tissue invasion. Its chymotrypsin-likeproteinase (CTLP) can degrade a number of basement membrane componentsin vitro, thus suggesting a contribution to tissue invasion by thespirochete. Periopathogen survival is dependent upon evasion ofcomplement-mediated destruction. Treponema denticola, an importantcontributor to periodontitis, evades killing by the alternativecomplement cascade by binding factor H (FH) to its surface.

In the healthy subgingival crevice, Treponema denticola account for ˜1%of the total bacteria. With the progression of periodontitis, theabundance of oral treponemes increases dramatically and can reach 40% ofthe total bacterial population. Disease severity correlates specificallywith the outgrowth of Treponema denticola and other bacterial species ofthe red microbial complex.

Treponema denticolais is an oral spirochete and periopathogen thattransitions from low abundance in healthy subgingival crevices to highabundance in periodontal pockets. The T. denticola response regulatorAtcR harbors the relatively rare, LytTR DNA binding domain. LytTR domaincontaining response regulators control critical transcriptionalresponses required for environmental adaptation. The functionaldiversity of the proteins encoded by the putative AtcR regulon suggeststhat AtcR sits at the top of a regulatory cascade that plays a centralrole in facilitating T. denticola's ability to adapt to changingenvironmental conditions and thrive in periodontal pockets.

While most bacteria, including spirochetes, employ two componentregulatory (TCR) systems and cyclic nucleotides to regulate adaptiveresponses, certain embodiments of the present invention are directed tothe T. denticola genetic regulatory system and signaling mechanisms todecrease the growth and maintenance thereof in the oral cavity.

One general take-away from the present invention relates to how best toadopt practices that establish and retain and maintain oral health suchthat individuals do not suffer from the array of different maladies thatare now understood to be related, whether directly or indirectly, tooral health. For example, it is common for individuals to get theirteeth cleaned a few times a year. Upon such a cleaning procedure,however, the dental surfaces are relatively “clean” of the biofilms thatwhere established thereon since the last dental cleaning visit. Insteadof proactively applying a beneficial composition of beneficial bacteriato the cleaned surfaces, however, it is common and typical practice tosimply have the dental patient leave the dental office, after schedulinganother 6 month visit, and thus leave the colonization of the dentalsurfaces up to the chance presence of bacteria that may then be presentin the person's mouth or surrounding environments. Given the growing andrecent knowledge of the nature of oral biofilms, populated by a myriadof bacteria of different but coexisting species of bacteria, it is oneaspect of the present invention to purposefully contact a person'srecently cleaned teeth with a composition that contains bacteriabelieved to be especially beneficial to the establishment of a “healthy”biofilm. This entails, in certain embodiments, a progressive andsuccessive contact of a person's teeth with different bacteria, with thestaging of contact with various bacteria based upon the knownsynergistic relationship between oral bacteria, and with the emphasisbeing to limit the most pathogenic bacteria known to cause some of theprevalent problems suffered by humans.

The limitation of the growth and establishment of a certain spirochete,namely, Treponema denticola, is a focus of various embodiments. The useof CRISPR-Cas and similar technologies to alter the genetic makeup ofsuch spirochete so as to lessen its infectivity in various regards isyet another way to accomplish this objective. Excision or retardation ofthe various virulence factors for this bacteria are other ways in whichsuch a goal can be achieved. Still other ways to accomplish thisobjective involves interfering with the admittedly complex interactionsand associations of other bacteria responsible for the growth ofspirochetes in the oral cavity. Thus, by directly addressing still othersupporting bacteria, one is able to indirectly, but nonethelesseffectively, limit the progression of spirochetes, and in particular,Treponema denticola, establishment and growth. By doing so, one is savedfrom the ravages of Alzheimer's disease, as well as the several otherdiseases that are noted as being related to the oral health of a person.Providing a tooth contacting substance at the time of a dental cleaningis preferred, as well as possible re-applications of compositions by theindividual so as to establish a preferred buildup of a beneficialbiofilm having particular bacteria constituents. Such formulations forbeneficial oral cavity health may vary dependent upon many factors, suchas the particular diet of the individual, the race of the individual,the age, etc. It is known that bacterial populations vary greatlybetween individuals, as well as within the same individual based onhealth and age. Thus, selection of particular compositions having apre-determined composition of bacteria formats and variety arecontemplated by the present invention.

Bacterial species are able to use various energy sources, includinglight and diverse organic and inorganic chemicals, for growth andmetabolism. These energy sources are used to produce an electrochemicalgradient that provides an electron donor for metabolism and allowsmaintenance of a membrane potential and proton motive force. Theenergetics of living systems are driven by electron transfer processesin which electrons are transferred from a substrate, which is therebyoxidized, to a final electron acceptor, which is thereby reduced. Incertain embodiments, it is possible to control metabolism by linkingbiochemical processes to an external electrochemical system, with suchlinking of biochemical and electrochemical systems permitting the use ofelectricity as a source of electrons for biotransformation reactions. Areversible biochemical-electrochemical link allows for conversion ofmicrobial metabolic and/or enzyme catalytic energy into electricity.

In still other embodiments, employment of technology described in U.S.Pat. No. 9,131,884 to Holmes is employed to achieve desired furthersteps to address communication of biological disease status to a thirdparty. For example, in certain embodiments, a medical device isassociated with a mucosal strip that comprises a microarray having abioactive agent capable of interacting with a disease marker biologicalanalyte and a reservoir having at least one therapeutic agent, with thedevice able to release the therapeutic agent(s) from the medical device.In certain embodiments, at least two microchips with a microarrayscanning device adapted to obtain physical parameter data of aninteraction between the disease marker biological analyte and thebioactive agent is employed. A biometric recognition device isconfigured to compare the physical parameter data with an analyteinteraction profile. The therapeutic agent releasing device controls therelease of the therapeutic agent from the reservoir. The interfacedevice facilitates communications between the microarray scanningdevice, biometric recognition device and the therapeutic agent releasingdevice. An energy source to power the medical device can take severalforms, including biologically activated batteries that are preferablyassociated with the strip.

In certain other embodiments, sugar is used as a source of energy,notably glucose that is converted into different sugars via an enzymaticcascade to provide necessary energy to create an electrochemicalgradient. This, in turn, is used to power an enzyme that synthesizesadenosine triphosphate (ATP). In contrast to natural catabolic pathwaysfor cellular glucose oxidation, a preferred embodiment does not rely onATP as an energy carrier. Instead, two redox enzymes oxidize glucose,generating reduced nicotinamide adenine dinucleotide (NADH) as the sugaris broken down. Another series of enzymes (as many as ten additionalenzymes) further breakdown the sugars and feed them back to the redoxenzymes to produce more NADH, with water and carbon dioxide being theonly by-products. NADH is a reducing agent and acts as an electronshuttle that carries electrons in living cells from one molecule toanother. NADH first transfers the electrons stripped from the glucose toa mediator with the help of an enzyme. The mediator then delivers theseelectrons to the battery's electrode, rendering it available to power anelectronic device. Such a battery mimics the way a living cell transferselectrons from one molecule to another to generate power, it runs onrenewable sugars, and has a high-energy storage density, rechargeableproviding an additional sugar solution. Malodextrin—a polymer made up ofglucose subunits—may be employed together with particular differentenzymes able to strip electrons from a single glucose molecule, thusharnessing the generated energy to power an electrical device.

To comply with written description and enablement requirements,incorporated herein by the following references are the following patentpublications: 2015/0216917 to Jones; 2015/0361436 to Hitchcock;2015/0353901 to Liu; 9,131,884 to Holmes; 2015/0064138 to Lu;2015/0093473 to Barrangou; 2012/0027786 to Gupta; 2015/0166641 toGoodman; 2015/0352023 to Berg.

While specific embodiments and applications of the present inventionhave been described, it is to be understood that the invention is notlimited to the precise configuration and components disclosed herein.Various modifications, changes, and variations which will be apparent tothose skilled in the art may be made in the arrangement, operation, anddetails of the methods and systems of the present invention disclosedherein without departing from the spirit and scope of the invention.Those skilled in the art will appreciate that the conception upon whichthis disclosure is based, may readily be utilized as a basis fordesigning of other methods and systems for carrying out the severalpurposes of the present invention to instruct and encourage theprevention and treatment of various human diseases. It is important,therefore, that the claims be regarded as including any such equivalentconstruction insofar as they do not depart from the spirit and scope ofthe present invention.

What is claimed is:
 1. A method of reducing the likelihood of migraineheadaches, comprising: providing to an individual in need thereof abuccal bioadhesive strip, said strip having a first and second side,said first side having a surface comprising a pattern defined by aplurality of spaced apart features each having at least one microscaledimension and having at least one neighboring feature having asubstantially different geometry, wherein an average spacing betweenadjacent ones of said features is between 0.5 and 5 .mu.m in at least aportion of said surface, the second side having a bioadhesive that isadapted to bind to a mucosal membrane for at least 1 hour while inside aperson's mouth, wherein said strip includes xylitol and at least oneencapsulated feature containing a bacteria selected from the groupconsisting of Lachnospira, Veillonella, Faecalibacterium and Rothia. 2.The method as set forth in claim 1, further comprising including on saidstrip an antibody that selectively inhibits the human calcitoningene-related peptide (CGRP) receptor.
 3. The method as set forth inclaim 1, further comprising including Lactobacillus spp. on said strip.4. The method as set forth in claim 1, further comprising administeringat least 6 g/fibre/day to the individual to increase the numbers ofbifidobacteria in the gut microbiome of the individual.
 5. The method asset forth in claim 1, further comprising including increasing in an oralcavity of the individual the number of both Bifidobacteriaceae andLactobacteriaceae bacteria.
 6. The method as set forth in claim 1,further comprising providing a high fibre diet that includes acetate. 7.The method as set forth in claim 6, wherein the amount of acetate issufficient to decrease the ratio of bacteria in the individual's oralcavity from the phylum Firmicutes compared to Bacteroidetes.
 8. Themethod as set forth in claim 1, further comprising includingEnterococcus faecium on the strip.
 9. The method as set forth in claim1, further comprising removing from the oral cavity of the individualgram negative bacteria associated with periodontitis, and within 2 hoursthereof, providing the individual with the strip as set forth inclaim
 1. 10. A method of reducing the likelihood of migraine headaches,comprising: providing to an individual in need thereof a buccalbioadhesive strip, said strip having a first and second side, said firstside having a surface comprising a pattern defined by a plurality ofspaced apart features each having at least one microscale dimension andhaving at least one neighboring feature having a substantially differentgeometry, wherein an average spacing between adjacent ones of saidfeatures is between 0.5 and 5 .mu.m in at least a portion of saidsurface, the second side having a bioadhesive that is adapted to bind toa mucosal membrane for at least 1 hour while inside a person's mouth,wherein said strip includes Lactobacillus reuteri bacteria to induce asustained induction of oxytocin, and providing the individual, prior tothe use of the strip, with an amount of antibiotic sufficient to reducethe number of undesired bacteria in the oral cavity said antibioticselected from the group consisting of tetracycline hydrochloride,doxycycline, and minocycline.
 11. The method as set forth in claim 1,further comprising retarding in the oral cavity of the individual thegrowth conditions for spirochetes and P. gingivalis.
 12. The method asset forth in claim 1, further comprising including Veillonella on thestrip.
 13. The method as set forth in claim 1, wherein said stripincludes at least one polymer selected from the group consisting ofpullulan, hydroxypropylmethyl cellulose, hydroxyethyl cellulose,hydroxypropyl cellulose, polyvinyl pyrrolidone, carboxymethyl cellulose,polyvinyl alcohol, sodium alginate, polyethylene glycol, tragacanth gum,guar gum, acacia gum, arabic gum, polyacrylic acid, methylmethacrylatecopolymer, carboxyvinyl polymer, amylose, high amylose starch,hydroxypropylated high amylose starch, dextrin, pectin, chitin,chitosan, levan, elsinan, collagen, gelatin, zein, gluten, soy proteinisolate, whey protein isolate, casein and mixtures thereof.
 14. Themethod as set forth in claim 1, wherein said strip includes at least0.2% xylitol by weight.
 15. The method as set forth in claim 1, whereinsaid strip further comprises bioluminescent material.
 16. The method asset forth in claim 1, wherein said strip is dissolvable in a person'smouth within a period of 1 hour.
 17. The method as set forth in claim 1,wherein the strip comprises at least one encapsulated feature thatcontains an agent selected from the group consisting of an antibiotic;lactic acid bacteria; and xylitol.
 18. The method as set forth in claim1, wherein said strip includes at least 200 mg of xylitol.
 19. Themethod as set forth in claim 17, wherein said agent further comprisesPrevotella bacteria.
 20. The strip of claim 1 wherein said strip has atleast one active ingredient encapsulated inside a frangible capsule thatmay be broken by the individual pressing against said strip with theindividual's tongue.